Artemisinins with improved stability and bioavailability for therapeutic drug development and application

ABSTRACT

A stable form of artemisinin wherein an artelinic acid or artesunic acid is complexed with cyclodextrin analogs, preferably, β-cyclodextrin. The complexed cyclodextrin artemisinin formulation shields the peroxide portion of the artemisinin backbone from hydrolytic decomposition rendering it stable in solution. Artelinic acid and cyclodextrin are placed into contact with one another to yield a 2:1 molecular species. Artesunic acid and cyclodextrin yield a 1:1 molecular species. The complexed cyclodextrin artemisinin formulation is effective for the treatment of malaria and is stable in solution for long periods of time.

[0001] This application claims priority of provisional application No.60/362,985 filed Mar. 7, 2002.

GOVERNMENT INTEREST

[0002] The invention described herein may be manufactured, used andlicensed by or for the U.S. Government.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] A novel form of artemisinins that are complexed with cyclodextrinfor solving stability problems associated with previous forms ofartemisinins.

[0005] 2. Brief Description of Related Art

[0006] Artelinic acid is an effective antimalarial agent when in contactwith the malarial parasite. However, artelinic acid has poor stabilityin solution and, thus, has limited bioavailability in vivo.Artemisinins, as a class, include such analogs as artelinic acid andartesunic acid among many others. Currently, no analog of theartemisinin class of compounds exists which can remain stable insolution. Injectable formulations of artemisinin analogs, such asartelinic acid and artesunic acid, are not FDA approved due to theirinstability in solution. All artemisinins contain a peroxide bridgesusceptible to hydrolytic cleavage. Artemisinins have been found toyield an inferior class of antimalarials due to these severe limitationsin chemical stability. Artemisinins are limited to only being packagedas solids for oral dosing, as previous patents have claimed. U.S. Pat.Nos. 6,326,023; 6,307,068; 6,306,896; 5,834,491; 5,677,331; 5,637,594;5,486,535; 5,278,173; 5,270,037; 5,219,865; 5,021,426; 5,011,951.

[0007] Application of an antimalarial formulation must be specific toadministration in hot, humid tropical regions native to the malarialparasite. Thus, chemical stability under drastic environmentalconditions is essential. Attempts to produce a more stable form ofartelinic acid have been accompanied by critical limitations. A solublesodium salt of artelinic acid has been successfully formulated, buteventually degrades over time. This is presumably due to a reformationof the insoluble acid. Numerous attempts at preventing this precipitatehave been unsuccessful.

[0008] The osmolality of the salt solution is significantly less thanthe predicted value indicating possible inter-molecular complexationthat may be responsible for eventual precipitation over time. Anamine-based buffer of artelinic acid has been successfully formulated,but yields a higher pH solution (>8.0) that induces significant veinirritation upon injection. Additional localized redness and swellingsurrounding the injection site is a notable contraindication to apreferred intravenous formulation. Additionally, amine-based buffershave been observed to take on a strong yellow hue over time. Themechanism of color formation has not been deduced, but implies amodification of the artelinate formulation, which is not conducive topharmaceutical preparations where a defined constant state of purity isessential.

[0009] U.S. Pat. Nos. 6,326,023; 6,307,068; 6,306,896; 5,834,491;5,677,331; 5,637,594; 5,486,535; 5,278,173; 5,270,037; 5,219,865;5,021,426; 5,011,951 are only directed to be packaged as solids for oraldosing.

[0010] Therefore, there is a need to provide a form of artemisinins thatsolve the stability problems associated with previous formulations.

[0011] It is an object of the present invention to provide a form ofartemisinins, such as but not limited to artelinic acid and artesunicacid that solves the stability problems associated with previousformulations.

[0012] It is another object of the present invention to provide a stableform of artemisinins that is injectable.

[0013] It is still another object of the present invention to provide astable form of artemisinins that does not develop a yellow hue overtime.

[0014] It is still another object of the invention to promotebioavailability and membrane permeability while decreasing thelikelihood of localized inflammation at the route of entry, thusincreasing its therapeutic activity.

[0015] These and other objects of the invention will become apparentupon a reading of the entire disclosure.

SUMMARY OF THE INVENTION

[0016] The present invention is directed to cyclodextrin complexed withartelinic acid or artesunic acid to form complexedcyclodextrin-artemisinin formulations in a 2:1 ratio of cyclodextrin perartelinic acid molecule or in a 1:1 ratio of cyclodextrin per artesunicacid molecule. The formulation is stable in solution, bioavailable,membrane permeable and does not cause inflammation upon injection.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1 is a plot of the hypsochromic shift observed withincreasing concentrations of cyclodextrin. Artelinic acidconcentration=10 mM.

[0018]FIG. 2a is an absorption spectrum of 10 mM artelinic acid with andwithout 1 mM β-cyclodextrin;

[0019]FIG. 2b is an absorption spectrum of 10 mM artelinic acid with andwithout 4 mM β-cyclodextrin;

[0020]FIG. 3 is a 600 MHz WATERGATE-TOCSY NMR spectrum of 1.2 mMartelinic acid with 2.5 mM β-cyclodextrin in PBS (pH 7.4);

[0021]FIG. 4 is a 600 MHz WATERGATE-ROESY NMR spectrum of 1.2 mMartelinic acid with 2.5 mM β-cyclodextrin in PBS (pH 7.4);

[0022]FIG. 5 is a 600 MHz WATERGATE-ROESY NMR spectrum of 1.2 mMartelinic acid with 2.5 mM β-cyclodextrin in PBS (pH 7.4);

[0023]FIG. 6 is a 600 MHz WATERGATE-ROESY NMR spectrum of artesunatewith an excess of β-cyclodextrin in PBS (pH 7.4);

[0024]FIG. 7a is the aromatic region of the 600 MHz proton spectra of1.2 mM artelinic acid;

[0025]FIG. 7b is the aromatic region of the 600 MHz proton spectra of1.2 mM artelinic acid complexed with 2.5 mM β-cyclodextrin in PBS (pH7.4);

[0026]FIG. 8a is the alkyl region of the 600 MHz proton NMR spectra of1.2 mM artelinic acid;

[0027]FIG. 8b is a 600 mHz proton NMR spectrum of 1.2 mM artelinic acidcomplexed with 2.5 mM β-cyclodextrin in PBS (pH 7.4);

[0028]FIG. 9a is a 600 MHz proton NMR spectrum of 2.5 mM β-cyclodextrinin PBS (pH 7.4);

[0029]FIG. 9b is a 600 MHz proton NMR spectrum of 2.5 mM β-cyclodextrinwith 1.2 mM artelinic acid in PBS (pH 7.4);

[0030]FIG. 10a is a 600 MHz proton NMR spectrum (protons number 2 to 6)of 2.5 mM β-cyclodextrin in PBS (pH 7.4);

[0031]FIG. 10b is a 600 MHz proton NMR spectrum (protons number 2 to 6)of 2.5 mM β-cyclodextrin complexed with 1.2 mM artelinic acid in PBS (pH7.4);

[0032]FIG. 10c is a 600 MHz proton NMR spectrum (protons number 2 to 6)of artesunate with an excess of β-cyclodextrin in PBS (pH 7.4);

[0033]FIG. 11 is a 600 MHz proton NMR spectrum of 2.5 mM β-cyclodextrinand 1.2 mM artelinic acid in PBS buffer at pH 7.4 with 1:9 D₂O/H₂O;

[0034]FIG. 12 is a 2D NOESY spectrum of 2.5 mM β-cyclodextrin and 1.2 mMartelinic acid in PBS buffer at pH 7.4 with 1:9 D₂O/H₂O;

[0035]FIG. 13 is a 600 MHz proton NMR spectrum of artelinic acid BNBP11387, WR# 255663;

[0036]FIG. 14 is a 600 MHz proton NMR spectrum of 2D TOESY spectrum of2.5 mM β-cyclodextrin and 1.2 mM artelinic acid in PBS buffer at pH 7.4with 1:9 D₂O/H₂O;

[0037]FIG. 15 is a 600 MHz proton NMR spectrum of 2D ROESY spectrum of2.5 mM β-cyclodextrin and 1.2 mM artelinic acid in PBS buffer at pH 7.4with 1:9 D₂O/H₂O;

[0038]FIG. 16 is a 600 MHz proton NMR spectrum of artesunate with anexcess of β-cyclodextrin in PBS buffer at pH 7.4;

[0039]FIG. 17 is a 600 MHz proton NMR spectrum of 2D ROESY spectrum ofartesunate with an excess of β-cyclodextrin in PBS buffer at pH 7.4;

[0040]FIG. 18a is the electrostatic potential map of the primary face ofβ-cyclodextrin looking into the molecule from the top;

[0041]FIG. 18b is the electrostatic potential map of the primary face ofβ-cyclodextrin as shown in FIG. 18a rotated to the left;

[0042]FIG. 18c is the electrostatic potential map of the secondary faceof β-cyclodextrin;

[0043]FIG. 18d is a molecular model of FIG. 18d illustrating thepositions of specific atoms;

[0044]FIG. 19a is a side view of the electrostatic potential map ofartelinic acid;

[0045]FIG. 19b is a rear view of the electrostatic potential map ofartelinic acid;

[0046]FIG. 20 is the electrostatic potential map of β-cyclodextrincomplexed with artelinic acid in a 2:1 molecular ratio;

[0047]FIG. 21 is a molecular model of β-cyclodextrin complexed withartelinic acid in a 2:1 molecular ratio showing degrees of insertion andinteraction between each molecule;

[0048]FIG. 22 is an axial view from the primary face of theelectrostatic potential map of β-cyclodextrin complexed with artelinicacid in a 2:1 molecular ratio indicating the electrostatic interactionbetween the benzoic acid moiety and one of the cyclodextrins;

[0049]FIG. 23 is a plot of osmolality versus concentration of artelinatein aqueous solution compared to theoretical determinations based on thecomplete disassociation of the salt;

[0050]FIG. 24 is a plot of osmolality versus concentration of alysine-artelinate salt preparation in aqueous solution compared totheoretical determinations based on the complete disassociation of thesalt;

[0051]FIG. 25 is a plot of osmolality versus concentration of alysine-artelinate salt preparation with 3 molar equivalents of lysine inaqueous solution compared to theoretical determinations based oncomplete disassociation of the salt;

[0052]FIG. 26 is the linear regression (R=0.994, p<0.0001) ofexperimentally measured osmolality of artelinate complexed withhydroxypropyl-β-cyclodextrin (1:2 mole ratio) in aqueous solution. Upperand lower 95% confidence intervals and 95% prediction limits are alsoindicated;

[0053]FIG. 27a-c are plots of relative deviation between experimentallymeasured osmolality and theoretical determinations based on completedisassociation for 3 aqueous artelinate formulations: lysine-artelinateprepared with 1 molar equivalent of lysine, lysine-artelinate preparedwith 3 molar equivalents of lysine, andhydroxypropyl-β-cyclodextrin-artelinate (2:1) complex;

DETAILED DESCRIPTION

[0054] The present invention is directed to a novel form of artemisininsthat remain stable over time in solution. The artemisinins may be, butare not limited to artelinic acid and artesunic acid. This novel form ofartemisinins uses a unique complexed form of the therapeutic agent withcyclodextrin analogs, such as but not limited to alpha-, beta-, andgamma-cyclodextrin analogs and their derivatives.

[0055] The present invention is directed to cyclodextrin complexed withartelinic acid in a 2:1 ratio which is a form of artemisinin that altersthe electron cloud surrounding the artemisinin molecule in such a way asto stabilize this agent to promote bioavailability and membranepermeability while decreasing the likelihood of localized inflammationat the route of entry. Thus, this form of artemisinin increases itstherapeutic activity. Artesunic acid was complexed with cyclodextrin,but in a unique 1:1 ratio in such a way as to stabilize the agent yieldsimilar increases in its therapeutic activity.

[0056] The stability of the artemisinins is achieved by changing thephysiocochemical properties such as but not limited to electron density,electrostatic potential and charge transfer mediated complexation.

[0057] The complexed cyclodextrin formulation of the artemisininsdescribed deliberately shields the peroxide bridge of the artemisininbackbone from hydrolytic decomposition. Additionally, the aromaticbenzoic acid portion of the artelinate molecule is also complexed with asecond cyclodextrin molecule. This unique 2:1 complexation withcyclodextrin is not intuitively obvious because artelinic acid alone isunstable in aqueous solution. Simply placing cyclodextrin in solutionwith artelinic acid would not achieve these results, as the artelinicacid would not be in contact with the cyclodextrin to form complexation.Futher, cyclodextrin is know to form complexes with itself and thus maynot be readily available in solution to interact efficiently andeffectively with the artelinic acid. The inventors have placed artelinicacid and cyclodextrin into contact with one another and have complexedthem in such a manner as to yield a stable 2:1 molecular species. Theinventors have also placed artesunic acid and cyclodextrin into contactwith one another and have complexed them in such a manner as to yield astable 1:1 molecular species.

[0058] The present molecules are stable under ambient or physiologicallyrelevant conditions.

[0059] Materials and methods

[0060] β-cyclodextrin was obtained from Sigma-Aldrich Corp., St. Louis,Mo. Artelinic acid was alkalinized with NaOH to yield the sodium salt.Standardized PBS buffer at a pH of 7.4 was obtained from InvitrogenCorp., Carlsbad, Calif.

[0061] Absorption Spectroscopy Studies.

[0062] Mixtures of artelinate (10 μM) were prepared with increasingconcentrations of β-cyclodextrin (0.0, 1.0, 4.0, 6.0, and 9.0 mM).Absorption spectra were collected on a Beckman DU Series 600Spectrophotometer.

[0063] The spectra collected indicated a clear hypsochromic or blueshift in the absorption maximum at 230 nm with increasing concentrationsof cyclodextrin. Hypochromic effects were also notable at 230 nm, aswell as the broader transitions observed at 275 and 382 nm (FIG. 1).This combined observation is consistent with inclusion interactions ofthe benzoic anion of artelinate with cyclodextrin.

[0064] Changes in observed isosbestic points at higher cyclodextrinconcentrations indicates a complicated molecular species containinggreater than a simple 1:1 molecular species (FIGS. 2a and 2 b). 1H NMRStudies.

[0065] Mixtures of β-cyclodextrin (2.5 mM) and artelinic acid (1.2 mM)were prepared in PBS (pH 7.4) and incubated at 37° C. for 2-3 hour topromote complexation prior to analysis.

[0066] All ¹H NMR data was collected using a Bruker DRX-600 spectrometeroperating at a proton frequency of 600.02 MHz at a temperature of 25° C.Solvent suppression was accomplished by application of the WATERGATE(WATER suppression by GrAdient Tailored Excitation) pulse sequencedeveloped by Sklenar and co-workers. This sequence provides excellentsuppression of the water resonance by a combination of rf pulses and aseries of gradient pulses. The sequence combines a non-selective 90°pulse with a symmetrical echo formed by two short gradient pulses inconjunction with a 180 selective (on water) pulse train.

[0067] The two-dimensional WATERGATE-TOCSY experiment employed amodified MLEV-17 spin-lock sequence for a total mixing time of 80 ms,including the 2.5 ms trim pulses at the beginning and the end of thespin-lock. The spectrum was collected with a spectral width of 7183.91Hz (11.972 ppm) using 2K data points with 32 scans per 256 t₁ incrementswith a 1.5 s recycle delay. The data was processed by multiplicationwith a 90° shifted sine-bell window function in each dimension, with onezero fill in the f₁ dimension before transformation to produce matricesconsisting of 512 data points in both dimensions.

[0068] The two-dimensional WATERGATE-NOESY spectra were collected with aspectral width of 7183.91 Hz (11.972 ppm) using 2K data points with 128scans per 512 t₁ increments with a 1.5 s recycle delay. The data wasprocessed by multiplication with a 90° shifted sine-bell window functionin each dimension, with one zero fill in the f₁ dimension beforetransformation to produce matrices consisting of 512 data points in bothdimensions. Two different experiments were conducted with mixing timesof 50 and 600 ms.

[0069] The two-dimensional WATERGATE-ROESY spectrum was collected with aspectral width of 7183.91 Hz (11.972 ppm) using 2K data points with 256scans per 512 t₁ increments with a 1.5 s recycle delay with a spin-lockmixing pulse of 400 ms. The data was processed by multiplication with a90° shifted sine-bell window function in each dimension, with one zerofill in the f₁ dimension before transformation to produce matricesconsisting of 512 data points in both dimensions.

[0070] Two-dimensional NMR methods were used to determine the degree ofcapping or complexation of artelinic acid by β-cyclodextrin. The 2DWATERGATE-TOSCY spectrum of artelinic acid (FIG. 3) clearly indicatesthat the individual spin-spin coupling networks of a mixture ofartelinic acid and β-cyclodextrin can be resolved. In FIG. 3, thespin-spin coupling network for β-cyclodextrin is shown at A and thespin-spin coupling network for the alkyl ring of artenilate is shown atB. The 2D-rotating frame NOE spectrum, WATERGATE-ROESY, of artelinicacid was collected at a mixing time of 400 ms and is shown in FIG. 4.The labeled intermolecular ROE interaction between the aromatic protonsof artelinic acid with both the anomeric and ring protons ofβ-cyclodextrin proves that this region of artelinic acid is complexedwith one molecule of β-cyclodextrin. In FIG. 4, A, B and C indicate theintermolecular dipolar ROE coupling between the aromatic protons ofartelinate with the glucose ring protons of β-cyclodextrin. The ROEbetween the meta protons are more intense than those observed for theortho protons indicating that meta protons are inserted deeper into thecavity. D and F indicate the dipolar coupling between the ortho protonsof artelinate with the two benzyl protons of artelinate. E indicates thedipolar coupling between the meta protons of artelinate with theanomeric protons of β-cyclodextrin. FIG. 5 shows the alkyl region ofthis same spectrum. The labeled intermolecular ROE's between the alkylring protons of artelinic acid with both the anomeric and ring protonsof β-cyclodextrin indicate that this region of artelinic acid iscomplexed with one molecule of β-cyclodextrin. These observations aresimilar to those reported by Nishijo (Nishijo, J.; Nagai, M.; Yasuda,M.; Ohno, E.; Ushiroda, Y. J. Pharm. Sci. 1995, 84, 1420-1426) and byRedenti (Redenti, E.; Ventura, P.; Fronza, G.;Selva, A.;Rivara,S.;Plazzi, P. V.; Mor, M. J. Pharm. Sci. 1999, 88, 599-607) in similarNMR β-cyclodextrin complexation studies. In FIG. 5, A represents aregion that contains the dipolar coupling between the ring protons ofβ-cyclodextrin and the alkyl ring proton of artelinate; and B representsthe region that contains the dipolar coupling of the anomeric protons ofβ-cyclodextrin with the alkyl protons of artelinate.

[0071] Two 2D WATERGATE-NOESY spectra were collected at mixing times of50 and 600 ms (data not shown). The NOESY spectrum collected at 600 msgave similar intermolecular and intramolecular NOE's to those observedin the ROESY spectrum, however the observed intensities were reduced.The NOESY spectrum collected at 50 ms did not exhibit the intermolecularNOE's between artenilate and β-cyclodextrin. This observation isconsistent with what one would expect due to the fact thatintermolecular NOE's require a longer mixing time to develop as comparedto intramolecular NOE's.

[0072] The 2D ROESY and NOESY data clearly indicate that both the alkyland aromatic regions of artelinic acid are complexed with one individualmolecule of β-cyclodextrin.

[0073] In FIG. 6, the spectrum of artesunate with an excess ofβ-cyclodextrin in PBS is shown. This data clearly indicates that theartesunate is capped by β-cyclodextrin in a 1:1 ratio. The region thatis represented by A contains the intramolecular dipolar coupling thealkyl ring proton of artesunate. The region that is represented by Bcontains the intermolecular dipolar coupling the alkyl ring proton ofartesunate with the ring protons of β-cyclodextrin. The region that isrepresented by C contains the intermolecular dipolar coupling the alkylring proton of artesunate with the anomeric protons of β-cyclodextrin.The region that is represented by D contains additional intramoleculardipolar coupling the alkyl ring proton of artesunate. The region that isrepresented by E contains the intramolecular dipolar coupling of theβ-cyclodextrin.

[0074]FIG. 7a shows the aromatic region of the 600 MHz proton spectra of1.2 mM artelinic acid and FIG. 7b is the aromatic region of the 600 MHzproton spectra of 1.2 mM artelinic acid complexed with 2.5 mMβ-cyclodextrin. Upon complexation the aromatic resonances of artelinateare both shifted upfield. The chemical shift values and the relativechanges in chemical shift values are given in Table 1. A similar shiftof aromatic protons resonances of ketoconazole on complexation withβ-cyclodextrin was reported by Redenti and co-workers (Redenti, E.;Ventura, P.; Fronza, G.;Selva, A.;Rivara, S.;Plazzi, P. V.; Mor, M. J.Pharm. Sci. 1999, 88, 599-607). In addition, the intensity of theresonance for protons 2 and 2′ is reduced indicating complexation. TABLE1 ¹H Chemical Shift Assignments (δ) for the Aromatic Protons and MethylProtons of Artelinic Acid Chemical Shift complexed with Proton ChemicalShift β-cyclodextrin Δδ (ppm) 3 and 3′ 8.09 7.82 +0.27 2 and 2′ 7.427.25 +0.17 methyl #1 0.98 1.02 −0.04 methyl #2 0.95 0.95 0.00

[0075]FIG. 8a shows the alkyl region of the 600 MHz proton spectra of1.2 mM artelinic acid and FIG. 8b shows 1.2 mM artelinic acid complexedwith 2.5 mM β-cyclodextrin. As seen from these spectra the chemicalshift position and the appearance of the methyl protons have changedindicating complexation of this region of the molecule withβ-cyclodextrin. The chemical shift of the resonances for methyl group #1are shifted upfield by 0.04 ppm (Table 1). The resonances for bothmethyl groups were broadened and less well resolved.

[0076]FIG. 9a is a 600 MHz proton spectra of 2.5 mM β-cyclodextrin andFIG. 9b is a 600 MHz proton spectra of 2.5 mM β-cyclodextrin with 1.2 mMartelinic acid. These spectra clearly indicate that chemical values forprotons 2 to 6 on β-cyclodextrin change on complexation with artelinicacid. Similar shifts in the proton resonances for β-cyclodextrin havebeen reported by Nishijo and co-workers (Nishijo, J.; Nagai, M.; Yasuda,M.; Ohno, E.; Ushiroda, Y. J. Pharm. Sci. 1995, 84, 1420-1426).

[0077]FIG. 10a-10 c show the proton spectra (protons number 2 to 6) of2.5 mM β-cyclodextrin, 2.5 mM β-cyclodextrin complexed with 1.2 mMartelinic acid and 1.2 mM artesunate in an excess of β-cyclodextrin,respectively. These spectra clearly indicate a different mode ofcomplexation for the two artemisinin analogs.

[0078] Table 2 summarizes the chemical shift assignments forcyclodextrin compared with the corresponding complexes with artelinicacid and artesunic acid as derived from FIGS. 9 and 10. The change inchemical shifts (Δδ) clearly demonstrate that both cyclodextrins of theartelinic acid complex and the cyclodextrin of the artesunic acidcomplex coordinate at the 3-H end or secondary face (FIG. 18) of thecyclodextrin. Further, the benzoic acid moiety of artelinic acidcoordinates deeply into the cyclodextrin pocket yielding significantchanges in chemical shift for the 3-H, 5-H, and 6-H protons. Incontrast, artesunic acid, which only binds to one cyclodextrin at theperoxide bridge, produced chemical shift changes of a lower magnitudeindicating a more shallow binding interaction. Lastly, for theartesunate-cyclodextrin complex the changes in chemical shift indicateΔ6 of 6H<5H<3H which clearly demonstrates this shallow bindinginteraction compared to the deep insertion of the benzoic acid moiety ofartelinic acid. This data clearly supports a unique stereochemicalarrangement based upon the physicochemical properties of each molecularspecies to yield a specific stable complex. TABLE 2 ¹H Chemical ShiftAssignments (δ) for the Cyclodextrin Protons (2 through 6) 2H 3H 4H 5H6H β-cyclodextrin 3.63 3.94 3.56 3.83 3.86 artelinic acid 3.61 3.83 3.533.72 3.74 Δδ 0.02 0.11 0.03 0.11 0.12 β-cyclodextrin 3.63 3.94 3.56 3.833.86 artesuate 3.62 3.88 3.55 3.79 3.84 Δδ 0.01 0.06 0.01 0.04 0.02

[0079]FIGS. 11 through 17 provide ancillary and supportive data that wasused in elucidating the structural conformation of the describedcyclodextrin complexes.

[0080] Molecular Electrostatic Potential Mapping and Docking/AffinityDeterminations.

[0081] Molecular Electrostatic Potential (MEP) maps on cyclodextrin andartelinic acid were developed by calculating electrostatic potentials onthe van der Waals surface of the molecules using the semi-empirical PM3molecular orbital theory as implemented in the SPARTAN software (SPARTANversion 4.0, Wavefunction, Inc., 18401 Von Karman Ave., #370, Irvine,Calif. 92715 U.S.A. 1995 Wavefuntion, Inc.). PM3 is a semi-empiricalquantum chemical theory model based on Thiel's integral formalismunderlying MNDO/d, and is used in conjunction with parameters for bothtransition and non-transition metals (reference: (a) W. Thiel and A.Voityuk, Theor. Chim. Acta., 81, 391, (1992); (b) W. Thiel and A.Voityuk, Int. J. Quantum Chem., 44, 807 (1992).

[0082] Molecular electrostatic potential (MEP) maps and theirelectrostatic potential energy isopotential profiles were generated andsampled over the entire accessible surface of a molecule (correspondingroughly to a van der Waals contact surface). The MEP maps provide ameasure of charge distribution from the point of view of an approachingreagent. This is calculated using a test positive charge as the probe.Thus, these types of profiles can provide an estimate of electronicdistribution surrounding the molecule so as to enable qualitativeassessment of any possible interaction with an approaching molecule.However, conformation search calculations using the “systematic search”technique via the single-point PM3 method of SPARTAN were used togenerate different conformers for each of the molecules. The minimumenergy conformer with highest abundance (a Boltzman population densitygreater than 70.0%) was chosen for full geometry optimization using thePM3 algorithm. The MEP profiles were generated on the optimized geometryof the molecules. The computations were carried out on a SiliconGraphics Octane workstation.

[0083] To further understand the binding affinities between cyclodextrinand artelinic acid, the complete optimized structures of both thecompounds have been considered and docking calculations using theDocking/affinity module in Insight II (Accelrys Inc., 9685 ScrantonRoad, San Diego, Calif. 92121-3752) were conducted. See Oprea, T. I. andMarshall, G. R. (1998) Receptor-based prediction of binding affinities.Perspectives in Drug Discovery and Design 9/10/11:35-61; and Insight IIUser Guide, San Diego: Accelrys Inc. (2002), which are hereinincorporated by reference.

[0084] Docking/affinity module in Insight II allows calculating thenonbonded energy between two molecules using explicit van der Waalsenergy, explicit electrostatic (Coulombic) energy, or both van der Waalsand electrostatic energies. The number of atoms included in thecalculation can be limited by specifying a monomer- or residue-basedcutoff. Other methods known in the art may be used, for example, thecomputation can be done using a pre-computed energy grid.

[0085] These molecular modeling determinations based on unique andspecific physicochemical properties of the artemisinins studiedcomplexed with β-cyclodextrin produced conceptual models which clearlyrationalized the direct physical measurements of the NMR experiments.FIGS. 18a-d illustrate the unique electrostatic potential map ofβ-cyclodextrin showing the primary binding faces (FIGS. 18a and 18 b)and secondary binding faces (FIG. 18c and FIG. 18d). Most notable is theunique net positive region 1 of the electron cloud at the primary face.

[0086]FIGS. 19a and 19 b illustrate the unique electrostatic potentialmap of artelinic acid. Most notable is the dense negative region 2 ofthe carboxylic acid tail as well as a more subtle negative region 3 ofthe peroxide bridge.

[0087]FIG. 20 clearly demonstrates the 2:1 complexation ofβ-cyclodextrin with artelinic acid. Two β-cyclodextrin molecules areshown at 4 and one artelinic acid molecule is shown at 5. The depth ofinsertion of the carboxylic acid tail compared to the peroxide bridgeportion of the molecule is more clearly illustrated in the correspondingball-and-stick model of the complex in FIG. 21 wherein twoβ-cyclodextrin molecules are shown at 4 and one artelinic acid moleculeis shown at 5.

[0088] Lastly, FIG. 22 directly illustrates the unique physicochemicalinteraction of the electrostatc potential map of cyclodextrin with thatof the artelinic acid tail. This axial view into the primary face of thesecond cyclodextrin molecule clearly illustrates this unique andselective electrostatic interaction. The negative region of theelectrostatic potential map is shown at 6 and the positive region ofelectrostatic potential map is shown at 7.

[0089] Simple docking calculations do not yield these results as theyassume an in vacuo environment. Inclusion complexes with cyclodextrinsare mediated by the release of high-energy water molecules from theinner core of the cyclodextrin molecule. Therefore, direct structuralmeasurements of the complex by techniques such as high resolutionmulti-dimensional NMR rationalized by physicochemical propertydeterminations such as but not limited to molecular electrostraticpotential mapping is specifically required to accurately characterizethese complexes.

[0090] Osmometry Determinations.

[0091] Solutions of hydroxypropyl-β-cyclodextrin and artelinic acid ofvaried compositions as indicated were measured at room temperature usinga Fiske ONE-TEN Osmometer (Fiske Associates, Norwood Mass., USA). Thesolvent for all experiments was ultra-pure distilled deionized water (18MΩ) filtered through a 0.45 μm filter. Small sample volumes (15 μL) weremeasured in units of mOsmol/kg water with an instrument repeatability of±2 mOsmol/kg water in the data range studied (0 to 400 mOsmol/kg water).The instrument was calibrated routinely with NIST standards of NaCl anda daily NIST reference of NaCl was verified at the start of each set ofexperiments.

[0092] Osmolality is a direct measure of the degree of moleculardissociation of a species in water. FIG. 23 illustrates the deviation ofmeasured osmolality in aqueous artelinate solutions versus theoreticalcalculations which assume complete dissociation. This deviation fromideality also appears to have a significant margin of error as observedby the marked degree of data scatter in the measurments.

[0093]FIGS. 24 and 25 illustrate a similar relationship between measuredosmolality and ideal dissociation with a lysine salt formulation and alysine salt formulation with 3 molar equivalents excess lysine. Allthree artelinate formulations appear to deviate strongly from ideality.Secondly, the measure of osmolality versus concentration of artelinateappears to be biphasic as demonstrated most clearly in FIG. 25, but alsoobserved in FIGS. 23 and 24.

[0094]FIG. 26 illustrates the strong linear correlation of theexperimentally measured osmolality of artesunate complexed withhydroxypropyl-β-cyclodextrin in aqueous solutions.Hydroxypropyl-p-cyclodextrin was chosen for all osmolalitydeterminations, as its aqueous solubility is greater than β-cyclodextrinand its well-established pharmacological compatibility for future i.v.drug formulations.

[0095] Measured deviation in osmolality of the artelinicacid-cyclodextrin (1:2) formulation after 28 days at room temperaturewas <7% in the concentration range of 15-25 mg/mL artelinate. This 7%deviation was consistently observed as an increase in osmolality due toan enhancement of solvation over time, rather than a decrease insolubility. The more concentrated solutions of cyclodextrin complexeswould need to incubate for longer periods of time to ensure maximumcomplexation.

[0096]FIGS. 27a-c illustrate the deviations from ideality of threeartelinate formulations, 1 molar equivalent of lysine shown at FIG. 27a,lysine-artelinate prepared with 3 molar equivalens of lysine shown atFIG. 27b and cyclodextrin-artelinate (2:1) complex shown at FIG. 27c.The artelinate-cyclodextrin formulation clearly deviates from idealityin a more predictable manner. The decrease in relative deviation withincreasing concentration is mostly likely due to enhanced complexationdue to a Le Chatelier's shift in solution equilibrium. This is notablycontrasted with the other two formulations which yield solutions thatdeviate in an increasing manner (10-15%) from 12 to 30 mg/mL.

[0097] Injectable formulation:

[0098] The stable form of artemisinin, the cyclodextrin complexed withartenilate in a 2:1 ratio, may be dissolved in saline, phosphatebuffered saline (PBS), deionized water or any other suitable aqueouscarrier for injection. The pH is preferably about 7.4. Generally, 40milligrams of artelinate complexed with cyclodextrin per milliliter ofsolution is suitable. A dose of about 4-6 mg of artelinic acid (incomplex) per kilogram of weight for a human is an appropriate dose. Aninjection of 10 ml of complex in solution or less is appropriate fortreatment.

[0099] The formulation of the cyclodextrin complexed with artelinate insolution can be prepared and pumped through a filter into an injectionvile, freeze dried for storage and later rehydrated with sterile wateror saline or PBS for injection. The cyclodextrin complexed withartelinate in solution can also be administered orally, sublingually, orin the form of a suppository.

[0100] Toxicity:

[0101] Cyclodextrins and artemisinins are both non-toxic to humans.However, large doses of cyclodextrins are not implicated in cases wherekidneys are not fully functional.

[0102] In Vitro Data

[0103] In Vitro Inhibition of Plasmodium falciparum.

[0104] See U.S. Pat. No. 6,284,772, which is herein incorporated byreference. The in vitro assays were conducted by using a modification ofthe semiautomated microdilution technique of Desjardins, et al. (1979)Antimicrob. Agents Chemther. 16:710-718 and Chulay et al. (1983) Exp.Parasitol. 55:138-146. Two strains of Plasmodium falciparum clones, fromCDC Indochina III (W-2), CDC Sierra Leone I (D-6). The W-2 clone issusceptible to mefloquine but resistant to chloroquine, sulfadoxine,pyrimethamine, and quinine. The D-6 clone is resistant to mefloquine butsusceptible to chloroquine, sulfadoxine, pyrimethamine, and quinine.These clones were derived by direct visualization and micromanipulationfrom patient isolates. Test compounds were initially dissolved in DMSOand diluted 400-fold in RPMI 1640 culture medium supplemented with 25 mMHEPES, 32 mM HaHCO₃, and 10% Albumax I (GIBCO BRL, Grand Island, N.Y.).These solutions were subsequently serially diluted 2-fold with a Biomek1000 (Beckman, Fullerton, Calif.) over 11 different concentrations. Theparasites were exposed to serial dilutions of each compound for 48 h andincubated at 37° C. with 5% 02, 5% CO₂, and 90% N₂ prior to the additionof [³H]hypoxanthine. After a further incubation of 18 h, parasite DNAwas harvested from each microtiter well using Packard Filtermate 196Harvester (Meriden, Conn.) onto glass filters. Uptake of[³H]hypoxanthine was measured with a Packard Topcount scintillationcounter. Concentration-response data were analyzed by a nonlinearregression logistic dose-response model, and the IC₅₀ values (50%inhibitory concentrations) for each compound were determined.

[0105]FIG. 28 indicates that both cyclodextrin formulations of artelinicacid (β-cyclodextrin and hydroxypropyl-β-cyclodextrin) yielded verysimilar in vitro activity against multi-drug resistant strains ofmalaria as indicated. All data indicated IC₅₀ concentrations within 4ng/mL of the uncomplexed artelinate salt (artelinic acid control).Therefore, complexation of the artemisinin molecule was not found toinhibit antimalarial efficacy.

[0106] Advantages

[0107] The complexed cyclodextrin-artemisinins formulation does notprecipitate or degrade over time. Formulations of artemisinins andcyclodextrin have been observed to remain completely soluble for up toseven weeks at elevated physiological temperatures (40 degrees C.)without any degradation and up to 6 months at room temperature. Thecomplexed cyclodextrin formulation of the artemisinins does not changecolor over time. Formulations of artemisinins and cyclodextrin have beenobserved to remain colorless for several weeks at elevated physiologicaltemperatures of 40 degrees C.

EXAMPLES Example 1 Formation of Artelinic Acid/Cyclodextrin Complex

[0108] Measure 2 moles of cyclodextrin and pre-dissolve in buffer,deionized water, or saline. Sonicate the mixture to completely dissolvethe cyclodextrin. Add 1 mole equivalent of artelinic acid and sonicate.Incubate at 40° C. for 2-3 hours. Higher concentrations of artelinicacid require longer incubation times, such as overnight, to promotecomplexation.

Example 2 Formation of Artesunic Acid/Cyclodextrin Complex

[0109] Measure 1 mole of cyclodextrin and pre-dissolve in buffer,deionized water, or saline. Sonicate the mixture to completely dissolvethe cyclodextrin. Add 1 mole equivalent of artesunic acid and sonicate.Incubate at 40° C. for 2-3 hours. Higher concentrations of artesunicacid require longer incubation times to promote complexation.

[0110] The use of the complexed cyclodextrin formulation of theartemisinins described provides a shielding effect to protect the bodyfrom local toxic effects from the antimalarial agent until the drug isdiluted sufficiently into the system. The process of making thecomplexed artemisinins of the invention can be performed on a largescale using similar conditions.

[0111] Having now fully described the invention, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theinvention as set for the herein.

What is claimed is:
 1. A composition comprising: cyclodextrin complexedwith artelinic acid in a 2:1 ratio.
 2. The composition of claim 1,wherein said cyclodextrin is coordinated in a manner so as to shield theperoxide bridge of the artemisinin molecule.
 3. The composition of claim1, wherein said cyclodextrin is selected from the group consisting ofalpha-cyclodextrin, beta-cyclodextrin and gama-cyclodextrin.
 4. Thecomposition of claim 1, wherein said cyclodextrin is beta-cyclodextrinselected from a group of beta-cyclodextrin analogs with similarcomplexing capabilities consisting of hydroxypropyl-beta-cyclodextrin,sulfobutyl ether-beta-cyclodextrin, andheptakis(2,6-di-O-methyl)-beta-cyclodextrin.
 5. The composition of claim1, further comprising an aqueous solution wherein said composition isdissolved in said aqueous solution.
 6. The composition of claim 5,having a pH of 7.4.
 7. The composition of claim 5, wherein said aqueoussolution is deionized water, saline solution, or phosphate bufferedsaline.
 8. A stable artemisinin formulation comprising:beta-cyclodextrin complexed with artelinic acid in a 2:1 ratio, whereinsaid one of said beta-cyclodextrin shields a peroxide portion of theartemisinin and a second of said beta-cyclodextrin is complexed with anaromatic benzoic acid portion of the artemisinin.
 9. The stableartemisinin formulation of claim 8, further comprising an aqueoussolution wherein said formulation is dissolved in said aqueous solution.10. The stable artemisinin formulation of claim 9, having a pH of 7.4.11. The stable artemisinin formulation of claim 9, wherein said aqueoussolution is deionized water, saline solution, or phosphate bufferedsaline.
 12. An antimalaria composition comprising: a complexedcyclodextrin formulation of artemisinin, wherein said cyclodextrin iscomplexed with artelinic acid in a 2:1 ratio in aqueous solution. 13.The antimalaria composition of claim 12, wherein said composition isstable in solution for up to 7 weeks at 40° C.
 14. The antimalariacomposition of claim 12, wherein said composition is bioavailable,membrane permeable and suitable for intravenous injection withoutirritability.
 15. The antimalaria composition of claim 12, wherein saidcomposition has a pH of about 7.4.
 16. The antimalaria composition ofclaim 12, wherein said complexed cyclodextrin formulation of artemisininremains in solution and does not precipitate with time.
 17. Theantimalaria composition of claim 12, wherein said composition is in aform of an intravenous dose, oral dose, sublingual dose, or suppository.18. A method of storing the antimalarial composition of claim 12comprising: filtering the antimalaria composition into a vile; freezedrying the composition in said vile to form a lyophilate, wherein saidlyophilate may be re-hydrated at a later date with an aqueous solutionfor injection.
 19. A method of treating a patient having malaria:comprising administering to said patient the composition of claim 12.20. The method of claim 19, wherein said administering is by intravenousinjection.
 21. The method of claim 19, wherein said administering is byoral dose, sublingual dose, or suppository.
 22. The method of claim 19,wherein said administering to said patient is by a dose of 4-6milligrams of artelinic acid per kilogram of body weight.
 23. The methodof claim 19, wherein said 40 milligrams of said artemisinin complexedwith cyclodextrin is dissolved per milliliter of aqueous solution.
 24. Amethod of making an artemisinin complex wherein cyclodextrin iscomplexed with artilinic acid in a 2:1 ratio that is stable in solutionand is suitable for injection and the treatment of malaria comprisingthe steps of: dissolving two moles of cyclodextrin in aqueous solutionto form a first solution; sonicating said first solution to dissolve thecyclodextrin; adding one mole of artelinic acid to said first solutionto form a second solution; sonicating said second solution; andincubating said second solution to form said stable artemisinin complexin solution.
 25. The method of claim 24, wherein said incubation isconducted at 40° C. for 2-3 hours.
 26. The method of claim 24, whereinsaid cyclodextrin is β-cyclodextrin.
 27. The method of claim 24, whereinsaid cyclodextrin is hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta-cyclodextrin or heptakis(2,6-di-O-methyl)-beta-cyclodextrin.28. The method of claim 24, wherein if concentrations of greater than10-15 mg of artelinic acid are used, incubation of said second solutionis conducted at 40° C. overnight.
 29. The method of claim 24, whereinsaid aqueous solution is selected from the group consisting of phosphatebuffered saline, saline solution and deionized water.
 30. The method ofclaim 24, wherein said stable artemisinin complex in solution is at a pHof 7.4.
 31. A method of making an artemisinin complex whereincyclodextrin is complexed with artesunic acid in a 1:1 ratio that isstable in solution and is suitable for injection and the treatment ofmalaria comprising the steps of: dissolving one mole of cyclodextrin inaqueous solution to form a first solution; sonicating said firstsolution to dissolve the cyclodextrin; adding one mole of artesunic acidto said first solution to form a second solution; sonicating said secondsolution; and incubating said second solution to form said stableartemisinin complex in solution.
 32. The method of claim 31, whereinsaid incubation is conducted at 40° C. for 2-3 hours.
 33. The method ofclaim 31, wherein said cyclodextrin is β-cyclodextrin.
 34. The method ofclaim 31, wherein said cyclodextrin is hydroxypropyl-beta-cyclodextrin,sulfobutyl ether-beta-cyclodextrin orheptakis(2,6-di-O-methyl)-beta-cyclodextrin.
 35. The method of claim 31,wherein if concentrations of greater than 10-15 mg of artesunic acid areused, incubation of said second solution is conducted at 40° C.overnight.
 36. The method of claim 31, wherein said aqueous solution isselected from the group consisting of phosphate buffered saline, salinesolution and deionized water.
 37. The method of claim 31, wherein saidstable artemisinin complex in solution is at a pH of 7.4.
 38. A methodof changing the physiochemical properties of artemisinin rendering itstable in solution, bioavailable, membrane permeable andnon-inflammatory comprising: adding cyclodextrin to artelinic acid underconditions to form a 2:1 complex wherein a peroxide portion of anartelinate backbone from the artelinic acid is shielded from hydrolyticdecomposition by a cyclodextrin and an aromatic benzoic acid portion ofthe artelinate is complexed with a second cyclodextrin.
 39. Anantimalarial composition comprising: a complexed cyclodextrinformulation of artemisinin, wherein said cyclodextrin is complexed withartesunic acid in a 1:1 ratio in an aqueous solution.
 40. A method ofstoring the antimalarial composition of claim 39 comprising: filteringthe antimalaria composition into a vile; freeze drying the compositionin said vial to form a lyophilate, wherein said lyophilate may bere-hydrated at a later date with an aqueous solution for injection. 41.The antimalarial composition of claim 39, wherein said composition isstable in solution for up to 7 weeks at 40° C.
 42. The antimalarialcomposition of claim 39, wherein said composition is bioavailable,membrane permeable and suitable for intravenous injection withoutirritability.
 43. The antimalarial composition of claim 39, wherein saidcomposition has a pH of about 7.4.
 44. The antimalarial composition ofclaim 39, wherein said complexed cyclodextrin formulation of artemisininremains in solution and does not precipitate with time.
 45. Theantimalarial composition of claim 39, wherein said composition is in aform of an intravenous dose, oral dose, sublingual dose, or suppository.46. A method of treating a patient with malaria: comprisingadministering to said patient the composition of claim
 39. 47. Themethod of claim 39, wherein said administering is by intravenousinjection.
 48. The method of claim 39, wherein said administering is byoral dose, sublingual dose, or suppository.
 49. The method of claim 39,wherein said administering to said patient is by a dose of 4-6milligrams of artesunic acid per kilogram of body weight.
 50. The methodof claim 39, wherein said 40 milligrams of said artemisinin complexedwith cyclodextrin is dissolved per milliliter of aqueous solution.
 51. Acomposition comprising: cyclodextrin complexed with artesunic acid in a1:1 ratio.
 52. The antimalaria composition of claim 51, wherein saidcyclodextrin is selected from the group consisting ofalpha-cyclodextrin, beta-cyclodextrin and gama-cyclodextrin.
 53. Thecomposition of claim 51, wherein said cyclodextrin is beta-cyclodextrinselected from a group of beta-cyclodextrin analogs with similarcomplexing capabilities consisting of hydroxypropyl-beta-cyclodextrin,sulfobutyl ether-beta-cyclodextrin,heptakis(2,6-di-O-methyl)-beta-cyclodextrin, to name a few.
 54. Thecomposition of claim 51, further comprising an aqueous solution whereinsaid composition is dissolved in said aqueous solution and is stable insaid aqueous solution.
 55. The composition of claim 54, having a pH of7.4.
 56. The composition of claim 54, wherein said aqueous solution isdeionized water, saline solution, or phosphate buffered saline.
 57. Astable artemisinin formulation comprising: beta-cyclodextrin complexedwith artesunic acid in a 1:1 ratio, wherein said beta-cyclodextrinshields a peroxide portion of the artemisinin.